1. Field of the Invention
This invention relates to methods and compositions for detecting nucleic acid sequences including deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) ["target nucleic acids" or "target nucleic acid sequence"] in a biological sample. The method permits the rapid determination of the presence of specific DNA or RNA sequences in the biological sample by hybridizing labeled DNA or RNA probes with the target nucleic acids in a solution. Following hybridization of the labeled probes to the target nucleic acid, the resulting hybridized and non-hybridized probes are separated by gel exclusion or affinity chromatography. The methods of this assay are suitable for use with biological samples, such as crude serum, plasma or tissue culture cells or tissue extracts or with purified or partially purified samples.
2. Description of the Prior Art
The nucleic acids DNA and RNA contain linear sequences of purine and pyrimidine bases which have the ability to form hydrogen bonds resulting in double-stranded helical regions. The process of forming a double-stranded hydrogen bonded helical region is referred to as hybridization or DNA renaturation. Such hybridization occurs when DNA or RNA samples containing complementary sequences are incubated under proper salt, pH and temperature conditions. Any two nucleic acid sequences can hybridize with one another, i.e., DNA:DNA, RNA:RNA or RNA:DNA, as long as they have a complementary nucleotide sequence. The rate of double helix formation is a function of both the concentration of reactants and the temperature. When one of the two nucleic acid chains contains a known sequence and a label, then the presence of a complementary nucleic acid sequence in an unknown solution can be determined by hybridization and detection of the label.
DNA probes labeled with radioisotopes have been widely used to localize specific nucleic acid sequences in mixtures of DNA restriction fragments fractionated by gel electrophoresis. A replica of a gel electrophoresis pattern is made by transferring fractionated DNA fragments to a sheet of nitrocellulose paper either by electrophoresis or by diffusion in a process called blotting. By hybridizing a radioactive probe to DNA or RNA bound to the nitrocellulose paper, hybrids are formed between the probe and the specific DNA sequence sought. Unhybridized probe is washed away and labeled areas of the nitrocellulose paper are detected as by autoradiography.
Another blotting method adapted to detect hepatitis B viral DNA in serum or plasma was described by Scotto et al., Hematology, 3:279-284 (1983). This method permits use of a larger sample volume of unextracted serum or plasma by binding sample DNA directly to nitrocellulose paper using a vacuum filtration apparatus.
The methods described above rely on binding the target nucleic acid sequences to a solid phase, such as nitrocellulose paper, then removal of unhybridized labeled probe by washing and detection of labeled probe hybridized with target nucleic acid sequences bound to the solid phase. These solid phase methods can result in extensive loss of sample, are time-consuming and involve numerous steps including solid phase preparation, binding, blocking and washing.
The procedures presently available for preparation of sample, hybridization and separation of hybridized probe from unhybridized probe are not generally suitable for routine use due to either the complexity of the procedures requiring highly trained personnel, the time-consuming nature of the procedure or the need for specialized equipment. There has been a need for a simple method which shortens the time required to perform the assay, requires a minimum quantity of materials, is reliable and reproducible and provides a method of quantitation.
Nobrega, et al., Analytical Biochemistry, 131:141-145, (1983) describe a method for detection of specific RNA transcripts during RNA processing wherein both the target nucleic acid and probe are in solution. In this method the free DNA probes are separated after hybridization utilizing electrophoretic migration on agarose and visualization by autoradiography.
The principle of separating small molecules from larger, higher molecular weight molecules based on differences in weight and/or conformation is known in the literature. Such methods include differential centrifugation, chromatographic methods, electrophoretic methods, dialysis and like methods.
Methods for the production of DNA containing the sequence of the hepatitis B virus genome by the cloning of the hepatitis sequence onto a bacterial plasmid are described in U.K. Patent Application No. GB 2034323A, published Jun. 4, 1980. Reagents and methods for the quantitative determination of viral DNA, such as hepatitis B virus DNA, utilizing a known amount of a phage vector was described in PCT Application No. WO 85/3951, published Sep. 12, 1985. Methods and compositions useful for detecting denatured, solid phase-affixed pathogen DNA, together with a labeled DNA probe suitable for hybridization was also described in U.S. Pat. No. 4,358,535.